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Glycodelin: A Major Lipocalin Protein of the Reproductive Axis with Diverse Actions in Cell Recognition and Differentiation

Department of Obstetrics and Gynecology (M.S., H.K., R.K.), Helsinki University Central Hospital, Haartmaninkatu 2, 00029 HUS, Helsinki, Finland; Department of Obstetrics, Gynecology and Reproductive Sciences (R.N.T.), University of California, San Francisco, California 94143-0132; and Laboratoire d’Hormonologie et de Biologie Moleculaire (E.M.), Institute National de la Santé et de la Recherche Médicale Unité 135, Hormones, Genes et Reproduction, Hopital de Bicetre, 94275 Le Kremlin-Bicetre, France

Correspondence: Address all correspondence and requests for reprints to: Markku Seppälä, Pihlajatie 20 B 15, 00270 Helsinki, Finland. E-mail: mseppala{at}pp.htv.fi

	Abstract

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
Glycodelin is a glycoprotein that belongs to the lipocalin superfamily. Depending on glycosylation, glycodelin appears in various isoforms. In the uterus, glycodelin-A is the major progesterone-regulated glycoprotein secreted into uterine luminal cavity by secretory/decidualized endometrial glands. The other tissues expressing glycodelin include fallopian tubes, ovary, breast, seminal vesicle, bone marrow, and eccrine glands. Glycodelin-A potently and dose-dependently inhibits human sperm-egg binding, whereas differently glycosylated glycodelin-S from seminal plasma has no such effect. Absence of contraceptive glycodelin-A in the uterus during periovulatory midcycle is consistent with an open "fertile window." Glycodelin induced by local or systemic administration of progestogens may potentially reduce the fertilizing capacity of sperm in any phase of the menstrual cycle. Glycodelin also has immunosuppressive activity. Its high concentration at the fetomaternal interface may contribute to protection of the embryonic semiallograft. Besides being an epithelial differentiation marker, glycodelin appears to play a role in glandular morphogenesis, as transfection of glycodelin cDNA into a glycodelin-negative breast cancer cells resulted in formation of gland-like structures, restricted proliferation, and induction of other epithelial markers. These various properties, as well as the chemistry, biology, and clinical aspects of glycodelin, continue to be areas of active investigation reviewed in this communication.

I. Introduction

II. The Glycodelin and Lipocalin Genes

III. Physicochemical Properties

A. mRNA

B. Protein

C. Folding

D. Glycosylation

E. Recombinant glycodelin

IV. Temporal and Spatial Expression

A. Uterus

B. Fallopian tubes

C. Ovary

D. Seminal plasma and seminal vesicles

E. Hematopoietic cells

F. Breast

G. Other tissues

V. Regulation of Synthesis

A. Estrogen

B. Progesterone, progestogens, and antiprogestins

C. Relaxin

D. Human chorionic gonadotropin (hCG)

E. Other

VI. Biological Activity

A. Effects on the immune system

B. Interaction with the gametes

C. Endometrial receptivity

D. Glycodelin as a differentiation-related glandular morphogen

E. Carrier functions

VII. Clinical Perspectives

A. Circulating glycodelin levels and ovarian function

B. Fertile window and contraceptive activity of the uterus

C. Fertility and infertility

D. Seminal plasma glycodelin and fertilization in vitro

E. Contraception

F. Pregnancy and pregnancy disorders

G. Endometriosis

H. Postmenopausal hormone replacement treatment (HRT)

I. Tumors

J. Immunosuppression and prevention of HIV transmission

VIII. Concluding Remarks and Future Directions

	I. Introduction

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
IN THE 1970s and 1980s, several investigators identified and/or isolated a protein in the human placenta, amniotic fluid, pregnancy decidua, and seminal plasma and, independently of each other, gave it various names according to the site of origin or physicochemical characteristics (see Table 1). Thus, names such as placental 2-globulin (1), chorionic 2-microglobulin (2), -uterine protein (3), placental protein 14 (PP14) (4), progestogen-dependent endometrial protein (PEP) (5), pregnancy-associated endometrial 2-globulin (2-PEG) (6, 7), human ß-lactoglobulin homolog (8, 9, 10, 11), and progesterone-associated endometrial protein (PAEP) (12) were suggested. The name PP14 was given on the basis of its purification from the human placenta that also contained the fetal membranes, the amnion, and the chorion (4). Parts of decidua are attached to the chorion, contaminating placental tissue, and this was found to be the source of the placental PP14 (13). In immunological tests, PP14 was found to be related to 2-PEG (14), PEP (15), and 2-chorionic 2-microglobulin (16). PEP, in turn, was found to be immunologically related to 2-uterine protein (17). The N-terminal amino acid sequence was first reported for PP14 (8, 9), disclosing similarity with ß-lactoglobulins from various species. The same sequence was reported for 2-PEG (10). The full primary structure of PP14 was first resolved from a decidual cDNA library (18), and essentially the same structure, with some splicing variants, was later confirmed for 2-PEG (19). Because PP14 was found to be synthesized in tissues other than endometrium and its molecular mass was not 14 kDa, the name PP14 appeared to be a misnomer.

	II. The Glycodelin and Lipocalin Genes

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
The glycodelin gene is 5.05 kb long and is divided into seven exons (Ref. 38 ; see Fig. 1). The size of the exons (or of their coding sequences) varies between 16 and 139 bp. The introns are also of a limited size (269–1371 bp). An ATACATAA sequence (considered as an equivalent of a TATA box) is localized 29 bases upstream from the cap site. Promoter-specific transcription factor-1 (Sp1)-like binding sites (GGCGGG) are present at positions -56 and -67 and in reverse orientation at -207 and -243. Four putative glucocorticoid/progesterone response elements (PREs) are found at positions -1799, -1071, -745, and -302 in the gene promoter, and two additional putative PREs are present at +1912 and +1965. Transcription is initiated 48 bp upstream from the first coding ATG of the glycodelin signal sequence.

View larger version (36K): [in this window] [in a new window]   Figure 1. Structure of glycodelin. Promoter region of the glycodelin gene (PAEP) is shown in the upper part. Numbering is relative to the transcriptional start site (the first base of exon 1 is number 1). ATG represents the translation-initiating codon. Sequences in the human glycodelin promoter with high degrees of homology to consensus cis-elements corresponding to PREs, cAMP responsive elements (CRE), activator protein-1 elements (AP-1), and Sp1 are indicated in the figure. Some of these sites have been mapped functionally by deletion or mutation of the wild-type promoter. [Adapted from C. Vaisse et al.: DNA Cell Biol 9:401–413, 1990 (38 ); R. N. Taylor and colleagues: Am J Obstet Gynecol 182:841–847, 2000 (68 ), and J Clin Endocrinol Metab 83:4006–4012, 1998 (69 ); and J. Gao et al.: Mol Cell Endocrinol 176:97–102, 2001 (92 ).] Splicing pattern of the glycodelin gene is shown under the diagram of promoter region. [Adapted from J. Garde et al.: Proc Natl Acad Sci USA 88:2456–2460, 1991 (19 ); C. Vaisse et al.: DNA Cell Biol 9:401–413, 1990 (38 ); and H. Koistinen et al.: Lab Invest 76:683–690, 1997 (45 ).] Exons encoding the mature protein are presented as gray boxes, 5' and 3' untranslated regions of exons as white boxes, and introns as lines. Part of exon 1 encoding the signal peptide (Sig.) is also shown. Splicing pattern presented under the figure represents the major glycodelin transcript. Codons encoding the Asn-28 and Asn-63 glycosylation sites are marked with arrows. Cystein residues are marked with asterisks. Swiss-Model-deduced tertiary structure of the glycodelin monomer is shown in the middle. [Adapted from H. Koistinen et al.: FEBS Lett 450:158–162, 1999 (57 ).] The S-S bridge is shown as a cylinder, and the atoms of asparagines of potential glycosylation sites (Asn-28, Asn-63, and Asn-85) are shown as balls (nitrogen as blue and oxygen as red). Representative examples of the major glycans in glycodelin-A and glycodelin-S are shown at the bottom. [Reproduced with permission from A. Dell et al.: J Biol Chem 270:24116–24126, 1995 (20 ), and H. R. Morris et al.: J Biol Chem 271:32159–32167, 1996 (44 )].

Use of somatic hybrid cells and of in situ hybridization allowed assignment of the glycodelin gene to chromosome 9q34 (39). This localization in the region of the ABO blood group locus was consistent with the observation made in bovines of a linkage between ß-lactoglobulins and the J blood group (homologous to the human blood group in this species). Indeed, sequencing of glycodelin cDNA showed 70% homology with the coding sequence of ovine ß-lactoglobulin (9, 18). At the chromosomal level, these similarities extended to the exon-intron organization: both genes contain seven exons, and in all cases the exons encode the same protein domains. The Human Genome Organization’s Gene Nomenclature Committee has decided that the official symbol for the glycodelin gene is PAEP (12).

After these initial observations, the DNA similarities were extended to a family of proteins referred to as lipocalins (48, 49, 50, 51). About 17 of these proteins, called kernel lipocalins, share 3 conserved sequence motifs. Most are secretory proteins. Several of these serve transport functions, binding hydrophobic ligands. Although the remaining lipocalins display low sequence similarity, they share a similar ligand-binding cleft, comprised of eight antiparallel ß-strands. The vertebrate lipocalin genes are characterized in most cases by a seven-exon/six-intron structure. Furthermore, most lipocalin genes are clustered at bands 33 and 34 on the long arm of human chromosome 9. In Mus musculus, these genes form clusters on syntenic regions of chromosomes 2 and 4. Lipocalins thus form a protein superfamily which, despite quite divergent primary sequence structure, shares a highly conserved folding conformation (49).

	III. Physicochemical Properties

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
A. mRNA
Glycodelin is encoded by a 900-bp mRNA, which is highly similar to those of ß-lactoglobulins of various species and other lipocalins. The highest similarity is in exon 2, which encodes the amino acid sequence involved in retinoic acid binding by ß-lactoglobulin. Single nucleotide polymorphism (39) and HinfI restriction enzyme polymorphism have been described, with a 5% frequency for allele A1 and 95% frequency for allele A2, in Finland (12). The latter has not been characterized any further to assess whether or not it represents a single nucleotide polymorphism.

Several splicing variants of glycodelin mRNA have been found in the female and male reproductive tracts (19, 45) and in hematopoietic cells of the megakaryocytic lineage (52). Some of the variants lack the coding sequences of the glycosylation sites and/or Thr-Asp-Tyr sequence, which is usually found in proteins of the lipocalin family (53).

B. Protein
Glycodelin is secreted from the decidua into amniotic fluid, which is an excellent source for purification (54, 55). In SDS-PAGE, the amniotic fluid glycodelin has a molecular mass of 28 kDa, and when studied by gel filtration, glycodelin is reported to behave as a homodimeric complex with a molecular mass of 50–60 kDa (4, 5, 7, 56). Based on molecular cloning and sequencing of its cDNA (18), glycodelin contains 180 amino acids, 18 of which correspond to the putative signal peptide. The predicted molecular mass of the mature polypeptide is 18,787 Da. The 162-residue sequence of glycodelin has extensive similarity to ß-lactoglobulins of various species. There are four cystein residues (positions 66, 106, 119, and 160) responsible for intramolecular disulfide bridges in ß-lactoglobulins, and they all are conserved in glycodelin. ß-Lactoglobulins contain no carbohydrate, whereas glycodelin has three potential N-linked glycosylation sites, at Asn 28, Asn 63, and Asn 85 (18).

An immunoreactive form of glycodelin was detected in seminal plasma two decades ago (2, 4, 32, 43). In isoelectric focusing, the isoelectric point (pI) of seminal plasma glycodelin (5.2–5.4) is higher than that of amniotic fluid glycodelin (4.6–4.9). Its molecular mass differed slightly from that of amniotic fluid glycodelin-A (43). Whereas the molecular weight and isoelectric points of these two glycodelin isoforms are different (32, 43), they have identical tryptic peptide profiles and immunoreactivity, and their primary protein structure was the same. Seminal plasma also contains smaller immunoreactive forms of the glycodelin protein. Some of them are probably posttranslational cleavage products (43, 45).

C. Folding
Differentially glycosylated glycodelin isoforms share similar thermodynamic parameters of reversible denaturation, suggesting that native folding of these isoforms is not influenced by the differences in glycosylation. The tertiary structure of glycodelin was predicted using an automated Swiss-Model service that extrapolates the conformation of a target sequence from the known three-dimensional structure of related family members. The Swiss-Model-deduced tertiary structure of glycodelin was found to be similar to that of bovine ß-lactoglobulin and other lipocalins (57). The circular dichroism spectrum of glycodelin was also similar to that of ß-lactoglobulin (57, 58, 59). Notably, an important difference between the structures of ß-lactoglobulin and glycodelin is in their glycosylation. Despite these structural similarities, the amino acid sequence of ß-lactoglobulin does not contain any of the glycosylation sites present in glycodelin.

D. Glycosylation
Glycodelin contains 17.5% carbohydrate (4). The observed charge differences in the isoelectric points of amniotic fluid glycodelin-A and seminal plasma glycodelin-S suggested differences in glycosylation, because after enzymatic deglycosylation and desialylation, these two glycodelin isoforms were indistinguishable from each other on SDS-PAGE and isoelectric focusing (43). The difference in glycosylation was confirmed by lectin binding studies showing that, unlike amniotic fluid glycodelin-A, seminal plasma glycodelin-S does not react with lectins from Wisteria floribunda or Sambucus nigra. These lectins react with GalNAc and NeuAc2–6GalNAc oligosaccharide sequences, respectively, present in glycodelin-A but absent from seminal plasma glycodelin-S.

The molecular weights of the glycodelin isoforms isolated from pregnancy serum, midterm and term amniotic fluid, first trimester decidua and term decidua, and secretory endometrium have been found to be identical (R. Koistinen, H. Koistinen, and M. Seppälä, unpublished observation). The glycodelin isoforms from all these female sources have similar isoelectric points, identical immunoreactivity, and they all react with W. floribunda and S. nigra lectins, indicating the presence of GalNAc and NeuAc2–6GalNAc oligosaccharide sequences. This suggests that all these glycodelin isoforms in female reproductive tract and serum are similarly glycosylated and different from male glycodelin-S.

In the Swiss-Model-deduced tertiary structure of glycodelin, the glycans are located in a way that would allow them to form a clustered saccharide patch (60), i.e., carbohydrates from more than one glycosylation site could form a cluster. Because the folding patterns of glycodelin-A and glycodelin-S appear to be identical, these glycoproteins provide an excellent model to study the effect of differential glycosylation on the conformational stability and function of a given glycoprotein.

Of the three putative glycosylation sites at Asn 28, Asn 63, and Asn 85, only the first two are glycosylated both in glycodelin-A and glycodelin-S (20, 44). The definitive carbohydrate structure analyses of glycodelin-A and glycodelin-S by fast atom bombardment and electrospray mass spectrometry showed that these two glycodelin isoforms are glycosylated in a completely different fashion. These remarkable differences provide evidence for gender- and tissue-specific glycosylation that may play an important role in reproduction.

1. Amniotic fluid glycodelin (glycodelin-A).
The major nonreducing epitopes in the complex-type glycans are Galß1–4GlcNAc (lacNAc), GalNAcß1–4GlcNAc (lacdiNAc), NeuAc2–6Galß1–4GlcNAc (sialylated lacNAc), NeuAc2–6GalNAcß1–4GlcNAc (sialylated lacdiNAc), Galß1–4(Fuc1–3)GlcNAc (blood group Lewis^x), and GalNAcß1-(Fuc1–3)GlcNAc (lacdiNAc analog of Lewis^x) (Ref. 20 and Fig. 1). Oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae at their terminal ends have been reported to manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell receptor (61), and a biantennary N-linked oligosaccharide bearing Lewis^x has been reported to inhibit E-selectin-mediated adhesion (62). Because the latter fucosylated epitope is also expressed on glycodelin-A, it has been postulated (20) that the immunosuppressive effect of glycodelin is mediated via blocking of the selectin-like binding sites by this carbohydrate sequence. However, monocyte binding by glycodelin does not require glycosylation (63, 64).

2. Seminal plasma glycodelin-S.
Analysis of the N-glycans of glycodelin-S by mass spectrometry revealed that the major difference from glycodelin-A is that glycodelin-S contains no sialylated glycans (44). Moreover, the glycans in glycodelin-S are unusually fucose rich, and the major complex-type structures are biantennary glycans with Lewis^x and Fuc1–2Galß1–4(Fuc1–3)GlcNAc (Lewis^y)-type antennae. Lewis^y epitope is considered to be relatively rare in other human glycoproteins, although it has been observed in other seminal plasma-associated proteins. Interestingly, Lewis^y epitope has been associated with cancer and programmed cell death (65). Glycosylation in glycodelin-S is highly site specific, because the site at Asn-28 contains only high mannose structures, whereas Asn-63 carries only complex-type glycans.

E. Recombinant glycodelin
Glycodelin has been produced in Pichia pastoris and Escherichia coli (63, 66). Cells from Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) have been used to produce glycosylated recombinant glycodelin (67). Analyses by lectin immunoassays and fast atom bombardment mass spectrometry have shown that recombinant glycodelin from the CHO cells is devoid of any lacdiNAc-based complex-type oligosaccharide chains present in glycodelin-A (20), and most of the N-glycans in the CHO cell product are lacNAc-based complex-type glycans. Contrary to the CHO cells, the human HEK293 cells produce recombinant glycodelin that contains the same carbohydrate structures as in native glycodelin-A. This is possibly based on the activity of ß1-GalNAc-transferase enzyme present in the HEK293 cells but hardly detectable in the CHO cells (67). Cultured in high glucose-containing media, the human HEK293 cells are particularly suitable for the production of the A-type recombinant glycodelin, as lowering of the glucose concentration and the addition of glucosamine results in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae (67). Like glycodelin-A, recombinant glycodelin from the HEK293 cells reacts strongly with the W. floribunda lectin, whereas recombinant glycodelin from the CHO cells reacts only weakly, if at all.

	IV. Temporal and Spatial Expression

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
A. Uterus
1. Endometrium and uterine flushings.
Explants of human secretory endometrium and human pregnancy decidua synthesize glycodelin (13, 36), and monolayer cultures from early pregnancy decidua release glycodelin in tissue culture (70). Immunohistochemical staining has localized glycodelin to secretory endometrial glands (Fig. 2) and to the glandular epithelium of the decidua spongiosa (71, 72, 73). Except for the first days of menstrual cycle when glycodelin remains in basal glands, proliferative endometrium contains no detectable glycodelin. Studies by various groups (29, 71, 72) have shown that human endometrium contains no detectable glycodelin during the periovulatory midcycle. In a more recent study (74) employing accurate timing of ovulation, the first appearance of glycodelin in endometrium was observed as early as d 16 (LH +3) of the cycle. In most studies, the protein has been reported to appear in endometrial glands 4–5 d after ovulation, first in some glands, then gradually increasing so that 10 d after ovulation all the glands are strongly positive (72). This corresponds to the measured glycodelin concentration in endometrial tissue, increasing toward the end of an ovulatory cycle (36).

View larger version (63K): [in this window] [in a new window]   Figure 2. Tissues synthesizing glycodelins. A, Ovary, fallopian tube, and endometrial glands. B, Localization of glycodelin in secretory endometrium by immunohistochemical staining. C, In situ hybridization of glycodelin mRNA in secretory endometrium. D, Tissues and cells in which glycodelin synthesis has been demonstrated.

During human pregnancy, the glycodelin concentration in decidualized endometrium and amniotic fluid is highest at 10–18 wk (see Fig. 3; Refs. 33 and 41). Conclusive evidence for synthesis by the endometrium comes from the demonstration of glycodelin mRNA in secretory/decidualized endometrium, as well as from the studies on incorporation of labeled precursor amino acids into immunoreactive glycodelin in cultured endometrium explants (18, 36, 37) and in isolated endometrial epithelial cells (69, 78). Similar studies on the placenta have given negative results (18, 36).

View larger version (28K): [in this window] [in a new window]   Figure 3. Glycodelin concentrations in women. A, Serum with respect to ovulation and progesterone secretion. B, Endometrium and amniotic fluid.

B. Fallopian tubes
Given that the fallopian tubes and the uterus are of Müllerian-tract origin, it is not surprising that glycodelin is present in fallopian tubes. The first study (82) addressed glycodelin concentration and localization at various phases of the menstrual cycle. Although no difference was observed in the glycodelin concentration between isthmic and ampullar parts of the tubes, cyclical variation in the glycodelin concentration was found in the fimbrial part, the concentration being higher in the secretory than in the proliferative phase of the cycle. A subsequent study (37) using in situ hybridization substantiated glycodelin synthesis in the fallopian tubal epithelium. Other investigators (84, 85) have confirmed these observations by demonstrating release of glycodelin from cells prepared from the mucosal layer, grown in monolayer from fimbrial, proximal ampullary, and distal ampullary regions of the human fallopian tube. Remarkable inter-individual variations have been observed, and there is no difference in the glycodelin production by the cells prepared from the proximal ampullary and distal ampullary parts of the tube from the same patient. However, cells from the fimbrial region appear to be more responsive to steroid stimulation of glycodelin secretion compared with the cells prepared from either the proximal or the distal ampullary regions (84), explaining the temporal changes observed in the first study (82).

C. Ovary
Glycodelin has been identified by immunoperoxidase staining in the normal human ovary (21). In the follicular phase, glycodelin is localized to areas of stromal cell condensation in ovarian cortex, theca interna, and the granulosa. In the luteal phase, glycodelin is stained in theca interna of the corpus luteum and luteinized granulosa cells, and also in the corpus albicans and Leydig cells of the ovarian hilus. However, these staining results were not correlated with the presence of glycodelin mRNA. In the rat ovary, glycodelin mRNA is restricted to granulosa cells (77).

The biological role of ovarian glycodelin is only beginning to be uncovered. In human folliculogenesis, glycodelin protein becomes detectable in granulosa and thecal cells in late secondary follicles. There is glycodelin in follicular fluid. However, the concentrations in follicular fluid and granulosa cells are low compared with those in amniotic fluid and decidualized endometrium. Importantly, only the luteinized granulosa cells, but not the cumulus cells, express glycodelin mRNA (86). The more restricted expression of the mRNA compared with protein results from glycodelin uptake by the cumulus cells, shown by experiments involving radiolabeled glycodelin. As glycodelin appears to bind on the acrosome area of the sperm (86), the role of cumulus cells in removing this glycodelin is an interesting possibility that is currently being investigated. Granulosa cells from ovarian disorders such as the polycystic ovary syndrome remain to be studied with respect to glycodelin synthesis.

D. Seminal plasma and seminal vesicles
The first identification of immunoreactive glycodelin in seminal plasma dates back to the 1970s (1, 2, 27) and is confirmed in many subsequent studies (4, 32, 34, 42, 45). The levels in vasectomized and nonvasectomized men are similar (32). Northern blot, in situ hybridization, and RT-PCR analyses showed that glycodelin mRNA is expressed in seminal vesicles and ampulla of the vas deferens, not in the testis, epididymis, or the prostate. Likewise, immunohistochemical staining and in situ hybridization have localized glycodelin to the epithelial cells and lumen of glands in the seminal vesicles and to the ampullary part of the vas deferens (45).

E. Hematopoietic cells
Contrary to mature red blood cells, erythroid precursors of human bone marrow cells contain immunoreactive glycodelin (22). Whereas untreated K562 leukemia cells do not contain glycodelin, treatment with tetradecanoylphorbol acetate (TPA), a differentiation stimulus, can induce strong expression of glycodelin mRNA and release of the protein from these cells (22, 52).

Two differentially spliced isoforms of glycodelin have been identified in conditioned medium of TPA-stimulated K562 cells (52). Both isoforms have also been detected by RT-PCR in two human megakaryocytic cell lines and in normal human megakaryocytes and platelets. The finding of hematopoietic glycodelin within the megakaryocytic lineage has been interpreted as an additional link between the coagulation, reproductive, and immune systems (52). Possible gender differences and regulation of glycodelin synthesis by progesterone in sites other than the female reproductive tract are obvious questions that remain to be elucidated.

F. Breast
The finding that glycodelin has significant sequence similarity with bovine ß-lactoglobulin (8, 9, 18), a normal constituent of whey, suggested that glycodelin is produced in the breast. Immunohistochemistry, Northern blotting, and RT-PCR analyses have been used to study glycodelin expression in normal breast tissue. As expected, glycodelin has been identified in ductal and lobular epithelium of normal breast tissue, and also in morphologically normal parts of the breast removed from breast cancer patients (47). The presence of glycodelin in normal breast tissue leaves open questions about the significance of glycodelin in breast cancer (see Section VII.I.3).

G. Other tissues
Immunohistochemical staining has been employed to study glycodelin in fetal, and adult nonreproductive tissues. No significant staining has been noted in any tissue in the fetus (87). In archival adult tissues, both glycodelin and its mRNA have been found in glandular tissues, e.g., in the lung and eccrine sweat glands (23). Glycodelin in these sites has not been characterized beyond immunoreactivity. Nevertheless, these findings demonstrate that none of the previously introduced tissue-specific names can correctly reflect all the sites of glycodelin synthesis.

	V. Regulation of Synthesis

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
A. Estrogen
Glycodelin synthesis and secretion are spatially and temporally regulated (Figs. 2 and 3). Proliferative phase endometrium does not contain glycodelin, and estrogens given for hormone replacement treatment (HRT) do not increase the circulating glycodelin concentration (11, 88). However, studies have indicated an association between follicular-phase serum estradiol levels and luteal-phase serum glycodelin concentration, supporting an effect of estrogen priming (80, 88, 89). More recently, a significant positive correlation has been found between serum estradiol concentration and endometrial glycodelin staining between d 12 and 24 in the natural cycle (73). However, transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is not estrogen dependent (69). The effect of estrogen on glycodelin transcription is not direct (68), but may be mediated by endometrial cell differentiation, e.g., via up-regulation of progesterone receptors.

B. Progesterone, progestogens, and antiprogestins
Several PREs have been identified in the glycodelin gene (38). This and the temporal expression in endometrium suggest that the synthesis of this glycoprotein is progesterone regulated. Glycodelin production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestogens in vitro (69).

Northern blotting, metabolic labeling, and fluorography have been used to assess glycodelin mRNA and protein synthesis in endometrial tissue and cells (68). Luciferase reporter constructs transfected into HeLa cells and endometrial adenocarcinoma cells (Ishikawa cells) have been used to determine whether progestogens could activate the glycodelin gene promoter. Progestogens stimulated glycodelin secretion in primary epithelial cell cultures. Glycodelin promoter-luciferase constructs expressing sequences 1100, 914, and 452 bp upstream of the transcriptional start site were sufficient to allow transactivation by promegestone (90), and transcriptional activity was dependent upon coexpression of progesterone receptors (PR). Here, progesterone receptor B (PRB), the predominant PR in secretory endometrial epithelium (91), was more stimulatory than progesterone receptor A (PRA) (68).

Basal glycodelin promoter activity has been localized to the region between -304 and +20 bp (92). This region contains three putative Sp1 binding sites. Mutation analysis at these sites has shown that two of them are active. In cells treated with medroxyprogesterone acetate, the promoter activity of glycodelin-luciferase construct increased 2.6- to 3-fold when cells were cotransfected with PRA or PRB. However, promoter activity was unchanged (90) or even slightly reduced (92) in cells treated with estradiol and cotransfected with estrogen receptor (ER) expression vector. Thus, whereas the effects of estrogen on glycodelin promoter function remain inconclusive, what is less controversial is that ligand-activated PRs stimulate glycodelin gene expression, mediated through the functional Sp1 sites. Curiously, this is not the first example of progestogen activation of an endometrial gene via Sp1 elements. In decidualized human endometrial stromal cells, progestogens increase the expression of Sp1 protein, which in turn appears to activate transcription of the tissue factor gene via overlapping Sp1 cis-elements in the gene promoter (93). Inhibitory interactions between PR complexes and Sp1 also have been observed and are postulated to reflect competition for the same transcriptional cofactors (94). On the basis of all the above findings, it can be concluded that glycodelin transcription, synthesis, and secretion by endometrial epithelial cells are stimulated by progesterone and progestogens.

The role of antiprogestins is intriguing. While having a stimulatory effect in some experimental settings in vitro (69) and a small, transient increase in serum levels after early pregnancy interruption (95), the antiprogestin mifepristone given to women with normal menstrual cycles in low daily doses brings about retarded endometrial histology and a significant decrease in endometrial expression of glycodelin (96).

In vivo studies also support the view that glycodelin secretion is associated with estrogen priming and progesterone action. Cyclical expression of glycodelin in endometrial tissue follows progesterone exposure (35, 36). After controlled ovarian hyperstimulation (COH) and natural cycle patients, endometrial glycodelin expression begins on cycle d 16 and increases as the luteal phase progresses (74). A significantly higher increase in glycodelin expression is found in COH cycles compared with natural cycles. From the onset, COH cycles show more glycodelin localization in a larger proportion of endometrial cells compared with natural cycles, and this increase is highly correlated with advancement of endometrial morphological dating (74). Increased expression of glycodelin in the endometria of COH cycles may be secondary to increased sex steroid and receptor levels.

A double-blind placebo-controlled study showed that micronized progesterone given over the luteal phase to women with unexplained infertility is accompanied by elevated serum glycodelin levels (97), and postmenopausal women taking estrogen-progestogen replacement treatment have elevated serum glycodelin levels at the end of progestogen treatment (11, 88). However, studies on explants from early pregnancy decidua have shown no increased glycodelin production when cultured in medium supplemented with progesterone (98). Here, prior in vivo exposure of decidual tissue to endogenous progesterone and human chorionic gonadotropin (hCG) may be confounding factors, so that high endogenous concentrations result in the relative insensitivity of decidual tissue to respond to the same exogenous hormones.

To determine whether subnormal glycodelin levels could be improved by progesterone treatment used to correct endometrial defects, correlation between serum glycodelin levels and histological maturation of endometrial biopsies taken during late luteal phase has been evaluated (99). Poor correlation has been found between serum glycodelin levels and histological maturation in endometrial biopsies, and there is no significant correlation between glycodelin levels and midluteal phase progesterone and estradiol. Moreover, no statistically significant differences in glycodelin values have been found depending on whether progesterone or any fertility drug is taken or not. However, such clinical observations cannot be taken as evidence against regulation of glycodelin secretion by progesterone because glycodelin secretion follows progesterone secretion by 3–4 d, explaining the lack of correlation between serum progesterone and glycodelin concentrations. Evidence shows that there is no rise in serum glycodelin concentration unless the serum progesterone rises first (35). Interestingly, in a normal ovulatory cycle, the administration of progestogens can actually decrease endogenous progesterone secretion (100), so the net effects of progestogens plus endogenous progesterone may not be as strong as expected. It is concluded that both progesterone and progestogens can be considered as regulators of glycodelin secretion in the uterus, whereas the effects of progesterone on glycodelin synthesis in sites other than the uterus remain to be elucidated.

C. Relaxin
In conception and nonconception cycles, profiles of serum relaxin and glycodelin concentrations are closely correlated, with the onset of relaxin secretion preceding that of glycodelin by 1–2 d (101). Relaxin is absent in the circulation of women without functional ovaries who become pregnant through donated embryo transfer (102, 103). Likewise, the glycodelin levels are also low or undetectable (104, 105, 106). These studies raise the possibility that relaxin may regulate glycodelin synthesis or secretion.

Published studies report discordant results on this aspect. Isolated human endometrial glandular epithelial cells have been cultured either with or without added porcine relaxin for up to 4 d (78). Cells incubated with relaxin increase the glycodelin production rate 2- to 6-fold and, as determined by solution hybridization/ribonuclease protection assay, the glycodelin mRNA concentrations increase 2- to 11-fold in cells incubated with relaxin, suggesting that relaxin activates glycodelin transcription. But this has not been found in all studies. Human relaxin has failed to stimulate de novo production of glycodelin and, in fact, relaxin has been found to repress progestogen-stimulated activation of the glycodelin promoter (68).

Two in vivo studies have also produced conflicting results. The first was a placebo-controlled study on the effect of intravaginal administration of human recombinant relaxin given for induction of labor at term pregnancy (107). Although there was a small increase in serum relaxin concentration, no difference was observed in serum glycodelin concentrations.

The second in vivo study using recombinant human relaxin injected to nonpregnant women for 28 d was also carried out in a double-blind and placebo-controlled fashion (101). Those women who demonstrated ovarian cyclicity showed sustained elevation of serum glycodelin levels during relaxin treatment, whereas those without ovarian cyclicity or placebo treated women showed no elevation. During relaxin administration, the elevation in glycodelin spanned over the whole menstrual cycle, including the periovulatory phase, when normally there is a nadir in serum glycodelin concentration. It is possible that, in a normal menstrual cycle, both luteal progesterone and relaxin are involved in the induction of endometrial glycodelin secretion. Progesterone may also stimulate endometrial relaxin synthesis (108, 109). The conflicting results of both in vitro and in vivo studies leave the conclusions on direct effects of relaxin on glycodelin synthesis and secretion open, because it is not known whether the increase in relaxin in any of the studies has been large enough to bring about a biological effect. Now that recombinant human relaxin is available, more clinical studies should become feasible to address the possible synergistic actions between progesterone and relaxin.

D. Human chorionic gonadotropin (hCG)
The patterns of the rise and the fall of circulating glycodelin and hCG concentrations are similar, the levels rising from implantation until pregnancy wk 10 and falling thereafter. Studies on explants of human secretory endometrium have failed to provide evidence for a stimulating role of hCG in endometrial glycodelin secretion (110), and similar results have been reported in studies on explants from early pregnancy decidua when cultured in medium supplemented with hCG (98). In baboons, between d 10 and 12 post ovulation, where the mid and apical regions of the endometrial glandular epithelium show a distinct punctate pattern, glycodelin increases between d 12 and 18 of pregnancy. The protein and mRNA expression was consistently higher in the deeper glands of the functionalis and basalis during early pregnancy. Exogenous hCG followed by estrogen and progesterone treatment in intact ovariectomized baboons up-regulated glycodelin expression between d 18 and 25 post ovulation, whereas estrogen and progesterone treatment in the absence of exogenous hCG did not increase the glycodelin synthesis (111). This would suggest that hCG acts in concert with estrogen and progesterone in increasing the glycodelin secretion. Analysis of uterine flushings from hCG-treated animals indicates that a minimum of 7 d of hCG treatment is required for glycodelin to be detectable in the uterine lumen.

E. Other
1. Clomiphene citrate.
Clinical studies on women undergoing stimulation of follicular growth with clomiphene citrate, followed by preovulatory hCG administration, have shown decreasing serum glycodelin concentrations during the administration of clomiphene (112). Here, clomiphene was given during the follicular phase, during which proliferative endometrium does not contain glycodelin. A direct glycodelin synthesis-reducing effect of clomiphene has been demonstrated in normally cycling young women undergoing tubal ligation (113). Reduced synthesis of glycodelin was found in endometrium 7–9 d after the urinary LH surge. The reduction was greater after larger doses of clomiphene (100 and 150 mg), indicating that the antiestrogenic effect of clomiphene citrate is reflected as reduced endometrial glycodelin secretion.

2. Tamoxifen and mifepristone.
Antiestrogen (tamoxifen) and antiprogesterone (mifepristone) appear to prolong the luteal phase when taken in combination (200 mg mifepristone and 40 mg tamoxifen) for 3 d starting on d LH +1 (114). The glycodelin levels were elevated when tamoxifen was given alone, but lower with combined treatment. Different modes of administration of these compounds show varying results. Thus, 5 mg mifepristone given once weekly did not inhibit ovulation, but it prolonged the follicular phase by 6–13 d and delayed endometrial development. This was associated with lower serum glycodelin levels (115). In another study (96), daily administration of 0.5 mg mifepristone significantly decreased endometrial expression of glycodelin. On the basis of these published studies, it is difficult to reconcile between the controversial findings with the two antiestrogens, clomiphene and tamoxifen, because the timing of drug intake relative to the normally occurring glycodelin secretion has been different.

As-yet-uncharacterized paracrine factors appear to play an important role in the regulation of glycodelin secretion from endometrial epithelial cells. Primary epithelial cells, but not stromal cells, secrete significant concentrations of glycodelin. However, when epithelial cells were placed in coculture with normal endometrial stromal cells embedded in a basement membrane extract (Matrigel, Collaborative Biomedical Products, Bedford, MA) substratum, glycodelin secretion by the epithelial cells was stimulated approximately 8-fold in vitro (116). As this effect was not observed in cocultures of endometrial stromal and epithelial cells grown on plastic, the data suggest that Matrigel can induce stromal factors to stimulate differentiation of the nearby epithelial cells. The factor(s) in Matrigel that can induce the epithelial cell-activating capacity of stromal cells have not yet been identified.

	VI. Biological Activity

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
A. Effects on the immune system
1. Fetomaternal defense mechanisms.
Implantation involves an intimate contact between cells from two genetically disparate organisms, the embryo (trophoblast) and the mother (decidua). Despite being a semiallograft, the human conceptus is not subjected to the laws of classical transplantation immunity. The major immune cells present at the implantation site are T cells, natural killer (NK) cells and macrophages. Macrophages and NK cells belong to the innate immune system—they require no prior exposure to antigen to react. Carbohydrate-lectin interactions are used by these cells to recognize foreign cells. B cells are absent or only few in number at the implantation site, confined to lymphoid aggregates in the basal layer (117, 118).

The first study (119) sparking research on immunosuppressive properties of glycodelin was based on observations that extracts of decidual tissue obtained from the first trimester of pregnancy showed potent immunosuppressive activity in mixed lymphocyte cultures (Table 3). This could be neutralized by treatment with a monoclonal antiglycodelin antibody. Purified glycodelin also exhibited in vitro immunosuppressive activity (119). Glycodelin-containing first-trimester decidual extract brings about a dose-dependent suppression of the mitogenic response to phytohemagglutinin (120), inhibits the production of IL-2 from mitogenically stimulated lymphocytes, leads to a reduced IL-2 receptor release (121), and reduces IL-1 production from mitogenically stimulated mononuclear cell cultures (117).

Glycodelin dose-dependently increases IL-6 production by epithelial cells prepared from secretory endometrium, with stimulated levels reaching twice the basal values (124), although glycodelin is less effective than IL-1 at stimulating IL-6 production. These results show that IL-6 production by human endometrial epithelial cells is stimulated by other immunomodulatory peptides and suggest that glycodelin may be part of the network of peptides in the endometrium that influences embryo implantation.

B. Interaction with the gametes
It is now firmly established that complex carbohydrates play a role in cell adhesion processes, including sperm-egg binding (132, 133, 134). Data about human sperm-egg binding can be obtained using an in vitro system, known as the hemizona assay (135). The method involves microbisection of the human egg, resulting in the generation of two equally matched hemispheres of the zona pellucida (hemizonae). By comparing the binding of fertile sperm in the presence and absence of a test substance, it is possible to quantitate the contraceptive effect of the test substance using this internally controlled system, without bringing about fertilization and an embryo. The hemizona assay has been used to test a number of different oligosaccharides, polysaccharides, and glycoproteins for their ability to inhibit human sperm-egg binding (134, 136, 137).

Glycodelin-A is the first endogenous glycoprotein that was found to potently and dose-dependently inhibit binding of human sperm to the zona pellucida (128). This effect appears to result from interaction between glycodelin and the sperm rather than between glycodelin and the oocyte. The inhibitory activity of glycodelin-A on sperm-egg binding is virtually complete at the concentrations reported for uterine tissue and uterine flushings during the midluteal phase of a normal menstrual cycle (36, 80, 81). Data show that glycodelin-A mediates this biological activity via its unusual oligosaccharide sequences that are not present in glycodelin-S from seminal plasma (Fig. 4 and Ref. 44).

View larger version (20K): [in this window] [in a new window]   Figure 4. Glycodelin-A inhibits human sperm-egg binding. [Reproduced with permission from H. R. Morris et al.: J Biol Chem 271:32159–32167, 1996 (44 ).] GdA, Glycodelin-A; GdS, glycodelin-S.

C. Endometrial receptivity
After fertilization in the fallopian tube, the zygote migrates to the uterus where it arrives around LH +4 and hatches. Implantation begins around d LH +5 with apposition of the hatched blastocyst to the luminal surface epithelium of the endometrium, followed by attachment, invasion, and anchorage of the trophoblast into the endometrial stroma (139). The limited period of uterine receptivity is estimated to span from d 18 to d 24 of a regular ovulatory cycle, i.e., from LH +5 to LH +11 (140, 141).

According to most studies, significant endometrial glycodelin secretion begins 4–5 d after follicle aspiration or ovulation, at LH +5–6 (80, 142), i.e., at the opening of the implantation window. Because glycodelin-A inhibits NK cell activity (123), monocytic cell chemotaxis (64), T cell proliferation (125), and induces T cell apoptosis (127) at the concentrations present in endometrial tissue and uterine fluid, it is likely that uterine glycodelin secretion plays an important role in the fetomaternal defense mechanisms (133). Interactions with the gametes on the one hand and with the immune cells on the other hand indicate that the recognition processes between immune cells and the gametes may have converged (20), whereas there appear to be differences with regards to the effects of glycosylation.

As compared with normal fertile women, patients with unexplained infertility have reduced concentrations of glycodelin in uterine flushings but not in plasma samples (143), suggesting that local glycodelin levels may play a role in uterine receptivity.

D. Glycodelin as a differentiation-related glandular morphogen
The glandular association of glycodelin expression is of particular interest because, besides in the endometrium, glycodelin has been found in glandular structures of many tissues including seminal vesicles, lobular and ductal epithelium of the breast, eccrine sweat glands, and parabronchial glands (Refs. 23 and 47 and Fig. 2). In view of its wide glandular expression, its role as a differentiation marker and in glandular morphogenesis has been addressed.

Under standard culture conditions, MCF-7 breast cancer cells do not express glycodelin. However, transfection of glycodelin cDNA into these cells causes dramatic changes in their growth behavior, with suppression of proliferation and formation of acinar structures (23). The transfected cells have lost their ability to grow on semisolid media because of apoptosis, and they exhibit up-regulated markers of organized epithelia, such as E-cadherin and cytokeratins 8 and 18. These observations suggest that glycodelin can induce epithelial differentiation. The other alterations in glycodelin cDNA-transfected cells include intracellular redistribution of ß-catenin. Both the parental and the glycodelin-transfected cells express the ß1 integrin subunit, whereas the parental MCF-7 cells do not express the collagen- and laminin-binding 2 integrin subunit. Interestingly, the 2 integrin appears on the surface of glycodelin-expressing cells, but the expression of another laminin-binding integrin, 6 subunit, is lost after glycodelin transfection. This is an unexpected finding, because the 6ß1 complex is a widely expressed laminin receptor in various glandular epithelia (144). Therefore, one laminin receptor appears to replace another after glycodelin expression. Taken together, the transfection of malignant cells with glycodelin cDNA can bring about changes resulting in less aggressive growth and more advanced differentiation, suggesting that glycodelin may play a role as a differentiation-related glandular morphogen.

E. Carrier functions
Glycodelin exhibits significant amino acid sequence similarity with ß-lactoglobulins that bind retinoic acid and retinoids (8, 9, 18, 145). Also the folding patterns of glycodelin and bovine ß-lactoglobulin are similar. However, glycodelin appears less stable than ß-lactoglobulin against thermal denaturation (57), and glycodelin is a glycoprotein, whereas ß-lactoglobulin is not.

Purified glycodelin-A has been used in retinoid binding experiments. Results by fluorescent quenching method show that, unlike ß-lactoglobulin, glycodelin does not bind retinoic acid or retinol (57). The reasons for this include the possibility that, despite considerable similarity in the overall folding patterns between glycodelin and ß-lactoglobulin, conformations of these proteins are different, as determined by their differences in the denaturation behavior (57).

	VII. Clinical Perspectives

Top Abstract I. Introduction II. The Glycodelin and... III. Physicochemical Properties IV. Temporal and Spatial... V. Regulation of Synthesis VI. Biological Activity VII. Clinical Perspectives D. Seminal plasma glycodelin... E. Contraception F. Pregnancy and pregnancy... G. Endometriosis H. Postmenopausal hormone... I. Tumors J. Immunosuppression and... VIII. Concluding Remarks and... References

 
A. Circulating glycodelin levels and ovarian function
The first quantitative data on glycodelin in serum of normally menstruating women showed the highest levels during the late luteal phase, when progesterone levels were declining (30). This has been confirmed in many studies, suggesting that late-luteal-phase serum glycodelin determinations may provide quantitative information regarding functioning corpus luteum and endometrial responsiveness to progesterone (30, 146, 147). Importantly, the serum glycodelin concentration rises in ovulatory cycles only (35) but, indeed, there is no correlation between serum glycodelin and progesterone levels. This is because the serum glycodelin level starts rising later than glycodelin becomes detectable in endometrial tissue and appears in uterine flushings. The glycodelin levels continue to rise until the onset of menstrual cycle over the period when progesterone levels decline. Despite the lack of a correlation in circulating levels, the association between glycodelin and progesterone is suggested by the observations that, in anovulatory cycles, both progesterone and glycodelin levels remain low throughout the menstrual cycle (see Fig. 3). As discussed below, the relationship between ovarian hormone production and local endometrial glycodelin synthesis shows better correlation than with peripheral serum levels.

In addition to the uterus, there are other tissues in which glycodelin synthesis has been demonstrated, and these may contribute to the circulating glycodelin pool. However, secretory and decidualized endometrium are by far the most important sources of the circulating glycodelin, demonstrated by the differences in levels between hysterectomized and nonhysterectomized postmenopausal women in response to HRT (11) and the marked elevation of serum glycodelin concentration during pregnancy (33), during which decidua and amniotic fluid have the highest concentrations (Table 2). Although their precise contribution remains to be investigated, in view of existing data, the contribution of nonreproductive tissues to serum glycodelin concentration is likely to be small.

B. Fertile window and contraceptive activity of the uterus
There is considerable variation in the timing of human fertilization during the menstrual cycle (148), mainly due to variation in the timing of ovulation. There is evidence that most fertilizations follow sexual encounters that have taken place during the 6 d that precede ovulation (149). The reasons for failure of fertilization during the postovulatory period are the lack of supernumerary ovulations and changes in the cervical mucus, hampering sperm motility. Other reasons may also exist, one of which may be glycodelin secretion. In a normal ovulatory cycle, secretion of glycodelin usually begins on the 5^th d after ovulation (70, 71, 72). Because of its inhibitory activity on sperm-egg binding, glycodelin-A may contribute to the contraceptive activity of the uterus during the latter half of the secretory phase (150, 151). This activity probably depends on the unique oligosaccharides present in glycodelin-A but absent from seminal plasma-derived glycodelin-S that has no contraceptive activity (44). It is possible that the absence of glycodelin-A is required for the fertile window to be open to allow the sperm to maintain their fertilizing capacity on their way to encounter the egg in the fallopian tube (150, 151).

C. Fertility and infertility
The initial studies describing cyclical changes of glycodelin in endometrial tissue raised legitimate optimism for the development of a biochemical test fo